We are studying the regulation of glutamine synthetase in mammals to provide a basis for detailed studies comparing mechanisms of enzyme regulation in normal and neoplastic cells. We have purified glutamine synthetase from rat liver to apparent homogeneity. The enzyme is sensitive to inhibition by L-glutamine and histidine when studied with Mn 2 ion, but not with Mg 2 ion. Alanine, glycine, inorganic phosphate, and glucosamine-6-phosphate also are inhibitors of the purified enzyme. Alphaketoglutarate and citrate, elsewhere reported to activate glutamine synthetase, were found to activate or inhibit catalytic activity depending on the concentration of divalent cation present; and by these and other criteria, appear to regulate activity by binding divalent cation required for activity. The apparent Km for L-glutamate changes substantially with the concentration of divalent cation in the assay solution, and the shape of the substrate saturation curve is likewise modified by the concentration of divalent cation present. The responsiveness of the enzyme to various inhibitors (e.g., alanine, histidine, and glutamine) is also dependent on the concentration of Mn used for assay. The ultraviolet absorption spectrum of the purified enzyme shows no evidence for bound nucleotide, as is found in the spectrum of adenylated E. Coli glutamine synthetase. Antibodies have been prepared using purified rat liver glutamine synthetase as antigen. The antibodies are free of other antibody activity against proteins in crude liver extracts in immunodiffusion studies. The antibody is being used to measure synthetic and degradative rates of enzyme protein in rats injected with very high specific activity amino acids. We have also studied glutamine synthetase in six Morris hepatomas. The enzyme specific activity in extracts of these hepatomas ranges from 5-40% of the activity found in host liver or in the liver of rats without hepatomas. Normal rats and tumor-bearing rats maintained on a protein-free diet have a two- to threefold decrease in activity. In comparison, extracts of hepatomas from rats on the protein-free diet demonstrate an increase in activity in liver extracts; extracts of hepatomas from rats on normal diets. Using the antibody developed in our laboratory, we performed immunotitration studies with HTC extracts. (Text Abridged.)